Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsid proteins (VP5 and VP7), within VIBs in co-transfected or infected cells. Further co-immunoprecipitation analysis confirmed that NS80 could interact with each core protein respectively. In addition, we found that newly synthesized viral RNAs co-localized with VIBs. Furthermore, time-course analysis of viral structural proteins expression showed that the expression of NS80 was detected first, followed by the detection of inner shell protein VP3, and then of other inner-capsid proteins, suggesting that VIBs were essential for the formation of viral core frame or progeny virion. Moreover, knockdown of NS80 by shRNA not only inhibited the expression of aquareovirus structural proteins, but also inhibited viral infection. These results indicated that NS80-based VIBs were formed at earlier stage of infection, and NS80 was able to coordinate the expression of viral structural proteins and viral replication.

Grass carp reovirus (GCRV) is a member of the genus Aquareovirus in the family Reoviridae, and contains five core proteins (VP1-VP4 and VP6) and two outer-capsid proteins (VP5 and VP7) in its particle. Previous studies have revealed that the outer-capsid proteins of reovirus are responsible for initiating infection, but the mechanism is poorly understood. Using baculovirusexpressed VP5 and VP7 to recoat purified cores, in vitro assembly of GCRV was achieved in this study. Recoated GCRV (R-GCRV) closely resembled native GCRV (N-GCRV) in particle morphology, protein composition and infectivity. Similar to N-GCRV, the infectivity of R-GCRV could be inhibited by treating cells with the weak base NH 4 Cl. In addition, recoated particles carrying an AsnAAla substitution at residue 42 of VP5 (VP5 N42A /VP7 R-GCRV) were no longer infectious. These results provide strong evidence that autocleavage of VP5 is critical for aquareovirus to initiate efficient infection.